Cyclic-glur6 analogs, methods of treatment and use

ABSTRACT

A composition which is reversible inhibitor of at least one neuron-specific PDZ domain comprising 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             R is a molecular transporter with or without a linker amino acid; 
             R 1  is at least about one amino acid covalently bound; and,
 
R 2  is isoleucine, leucine, alanine, phenylalanine, or valine, and methods of use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of, and claims priorityto, PCT/US2010/035188, filed on May 18, 2010, which claims the benefitof priority under 35 U.S.C. §119(e) to U.S. Patent Application No.61/179,055 filed on May 18, 2009, the disclosures of which areincorporated herein in their entirety by reference.

STATEMENT OF GOVERNMENT RIGHTS

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of RO1 DA018428awarded by NIH/NINDS (Funding & Research. NINDSExploratory/Developmental Grant), NIH/NIDA and NIH Grant No.5R21NS061176-02.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 17, 2010, is namedB0197006.txt and is 4,120 bytes in size.

FIELD OF THE INVENTION

A composition which is reversible inhibitor of at least oneneuron-specific PDZ domain comprising

wherein

R is a molecular transporter with or without a linker amino acid;

R₁ is at least about one amino acid covalently bound; and,

R₂ is isoleucine, leucine, alanine, phenylalanine, or valine, andmethods of use.

BACKGROUND OF THE INVENTION

The PDZ domain is a common structural domain of 80-90 amino-acids foundin the signaling proteins of bacteria, yeast, plants, and animals. PDZis an acronym combining the first letters of three proteins—postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor(DlgA), and zonula occludens-1 protein (zo-1)—which were firstdiscovered to share the domain. PDZ domains are also referred to as DHR(Dlg homologous region) or GLGF (glycine-leucine-glycine-phenylalanine)(SEQ ID NO: 1) domains. These domains have been reported as helping toanchor transmembrane proteins to the cytoskeleton and hold togethersignaling complexes.

There are roughly 260 human PDZ domains, though since several PDZ domaincontaining proteins hold several domains, the actual number of PDZproteins is closer to 180. Blazer et al., “Small MoleculeProtein-Protein Interaction Inhibitors as CNS Theraputic Agents: CurrentProgress and Future Hurdles,” (Neuropsychopharmacology, 1-16, (2008))(the teachings of which are incorporated herein by reference) statesthat “PDZ domains are important scaffolding components in many signalingsystems, with an extensive role in the development and maintenance ofboth pre- and post-synaptic structures. Development of reversible smallmolecule inhibitors that target neuron-specific PDZ domains wouldprovide useful tools to probe the many functions of these importantscaffolds.” In a drug discovery program, stable, selective, highaffinity chemical probes for PDZ domains will be useful in theidentification and quantification of cellular protein therapeuticcandidates having endogenous activity at the PDZ binding modules.Attention is drawn to Udugamasooriya et al., “Bridged PeptideMacrocycles as Ligands for PDZ Domain Proteins,” Organic Letters,7(7):1203-1206 (2005), the teachings of which are incorporated herein byreference.

It has been reported that glutamate is the most prominentneurotransmitter in human physiology as present in over 50% of nervoustissue. Without being bound by any particular theory, it is believedthat glutamate acts upon two classes of receptors, one containing aligand-gated cation pore, called ionotropic glutamate receptors and theother class that responds to glutamate by mediating second-messengerproteins, called metabotropic glutamate receptors. The ionotropicreceptors are further classified based upon preferential agonistbinding/activation by N-methly-D-aspartate (NMDA-receptors),AMPA-receptors and kainate-receptors. All three glutamate receptors arepermeable to sodium with the NMDA-R having a preference for calcium,and, when activated by glutamate, these ions enter the neuron throughthe central pore of the receptor, leading the neuron to depolarize.

Also noted are the following, the teachings of which are incorporatedherein by reference (as are all references cited in this document):

-   1. Li et al., “Thermodynamic profiling of conformationally    constrained cyclic ligands for the PDZ domain,”Bioorganic &    Medicinal Chemistry Letters, 14, 1385-1388 (2004)-   2. Guy et al. “Small molecule inhibition of PDZ-Domain interaction,”    U.S. Pat. No. 7,141,600.-   3. Chamila N. Rupasinghe and Mark R. Spaller, “The interplay between    structure-based design and combinatorial chemistry,” Current Opinion    in Chemical Biology, 10, 188-193 (2006).-   4. Sharma et al., “Design, synthesis, and evaluation of linear and    cyclic peptide ligands for PDZ10 of the multi-PDZ domain protein    MUPP1,” Biochemistry, 46, 12709-12720 (2007).-   5. Cui et al. “PDZ protein interactions underlying NMDA receptor    mediated excitotoxicity and neuroprotection by PSD-95    inhibitors,” J. Neuroscience, 27, 9901-9915 (2007).-   6. Klosi et al., “Bivalent peptides as PDZ domain ligands,”    Bioorganic & Medicinal Chemistry Letters, 17, 6147-6150 (2007).-   7. Saro et al. “A thermodynamic ligand binding study of the third    PDZ domain (PDZ3) from the Mammalian Neuronal Protein PSD-95,” NIH,    46, 6340-6352. (2008).-   8. Gomika et al. “A chemical library approach to organic-modified    peptide ligands for PDZ domain proteins: A synthetic, thermodynamic    and structural investigation,” Chem. Bio. Chem., 9, 1587-1589    (2008).-   9. D. Gomika Udugamasooriya and Mark R. Spaller “Conformational    constraint in protein ligand design and the inconsistency of binding    entropy.” Biopolymers, 89, 653-667 (2008).-   10. Tao Li, “Studies of ligand-protein interaction for the third PDZ    domain (PDZ3) of mammalian post-synaptic density-95 (PSD-95)” (Jan.    1, 2005). ETD Collection for Wayne State University. Paper    AAI3198697. http://digitalcommons.wayne.edu/dissertations/AA13198697-   11. Sutcliffe-Goulden et al., “Receptor-binding cyclic peptides and    methods of use,” U.S. Ser. No. 11/198,884 (2005).

Further mention is made of Goun et al. “Molecular Transporters:Synthesis of Oligoguanidinium Transporters and Their Application to DrugDelivery and Real-Time Imaging,” ChemBioChem, 7:1479-1515 (2006) andStewart et al., “Cell-penetrating peptides as deliver vehicles forbiology and medicine,” Org. Biomol. Chem., 6:2242-2255 (2008), TheHandbook of Cell PenetratingPeptides, Second Edition, Ülo Langel, Ed,CRC (2006), and Cell-Penetrating Peptides: Processes and Applications,Ülo Langel, Ed, CRC (2002).

Various methods for producing cyclic peptides have been described. Forexample, chemical reaction protocols, such as those described in U.S.Pat. Nos. 4,033,940 and 4,102,877, have been devised to producecircularized peptides. In other techniques, biological and chemicalmethods are combined to produce cyclic peptides. Some methods involvefirst expressing linear precursors of cyclic peptides in cells (e.g.,bacteria) to produce linear precursors of cyclic peptides and thenadding of an exogenous agent such as a protease or a nucleophilicreagent to chemically convert these linear precursors into cyclicpeptides. See, e.g., Camerero, J. A., and Muir, T. W., J. Am. Chem.Society. 121:5597 (1999); Wu, H. et al, Proc. Natl. Acad. Sci. USA,95:9226 (1998).

Also noted are Martin Linhult et al., “Evaluation of different linkerregions for multimerization and coupling chemistry for immobilization ofa proteinaceous affinity ligand,” Protein Engineering, vol. 16 no. 12pp. 1147-1152, Oxford University Press (2003).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the reaction between on-resin3-nitro-2-pyridinesulphenyl (Npys) protected cysteine-ologoarginine andunprotected cysteine containing macrocyclic ligand.

SUMMARY OF THE INVENTION

This invention comprises a composition which is reversible inhibitor ofat least one neuron-specific PDZ domain comprising

wherein

R is a molecular transporter with or without a linker amino acid;

R₁ is at least about one amino acid covalently bound; and,

R₂ is isoleucine, leucine, alanine, phenylalanine, or valine.

In some embodiments, R₁ is β-alanine. In particular embodiments, R₂ isvaline. It is contemplated that in some embodiments the P, position islysine, aspartic acid, glutamic acid or arginine. In a specificembodiment, the composition has the structure:

In some embodiments R comprises a liposome, steroid, polyamine,nanotube, nanoparticle, dendrimer, cell-penetrating peptide,protein-transduction domain amino acid, peptoid, (N-substitutedglycine), oligogcarbamate, arginine oligomer of about 6-20 units (SEQ IDNO: 2) D-arginine oligomer, spaced arginine oligomer, N-argininepeptoid, oligoarbamate transporter, or tetrameric dendrimer. In additionR may comprise (SEQ ID NO: 3)

The practice of this invention further contemplates a method oftreatment of neuro-stress comprising administering to a subject atherapeutically effective amount of the composition of Structure 5. Inspecific embodiments of the method of neuro-stress is selected from thegroup comprising stroke, traumatic brain injury, epilepsy, pain orneurodegenerative disease. It is particularly contemplated that themethod employs administration in advance of said neuro-stress and isalso contemplated for therapeutic intervention against further neuronaldamage from the neuro-stress. Further contemplated is cotreatment with atherapeutically effective amount of an NMDA receptor antagonists such asmemantine, ketamine, select NMDA-R2B antagonists or currentneuro-protective therapeutics such as mannitol. Cotreating agents areadministered before, after or at generally the same time as thecompositions of Structure 5.

The method of treatment encompasses therapeutic effective dose of thecomposition of Structure 5 from about 0.1 μM to about 100 andparticularly from about 20 μM to about 40 μM. Administration includesparenteral, oral, buccal, sublingual, or by nasal spray. Particular noteis made of intrathecal administration. When employing cotreatment withketamine, doses of from about 10 to about 250 mg are noted.

DETAILED DESCRIPTION OF THE INVENTION

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms are to be broadly construed also comprising amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an .alpha. carbonthat is bound to a hydrogen, a carboxyl group, an amino group, and an Rgroup, e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium. Such analogs have modified R groups (e.g., norleucine)or modified peptide backbones, but retain the same basic chemicalstructure as a naturally occurring amino acid. Amino acid mimeticsrefers to chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally occurring amino acid such asβ-alanine.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

For clarity,

indicates a alkyl chain with a number of carbon atoms, here 4, and aterminal amido group:

Structures of the present invention can be viewed as linear peptideswith a cyclic element. In Structure 1a (an annotated version ofStructure 1) the cyclic element is comprised of two amino acid“arms”—Arm 1 and Arm 2, joined by a “bridge” or “variable region”element, here beta-alanine (β-alanine), and amino acid, P⁻². In thisexample, Arm 1 is glutamic acid (the amino acid in the P⁻¹ position),Arm 2 is lysine (the amino acid in the P⁻³ position), along with theamino acid of P⁻² and the bridge element. The bridge element iscontemplated as comprising from a single amino acid to 6 or more aminoacids, though particular note is made of a the bridge element comprisinga single amino acid or two or three amino acids. Covalent linkage of thebridge element is particularly contemplated.

These cyclic elements can be formed of any amino acids offering greatvariability is size and side chain availability. These elements offerincreased stability against proteases, enhanced binding selectivity andenhanced ability to modify/select permeability.

Associated with this cyclic moiety is a segment to facilitate cell entryof the drug. This is termed an “Incorporation Sequence.” In someembodiments this includes from 1 to 5 additional amino acids as linkerelements linking the cyclic element to a transporter element. Moleculartransporters included an element known as a poly arginine tail,Structure 3. Incorporation Sequence is broadly defined to cover variousmolecular transporters that permit insertion into the endosome with orwithout linker elements. Molecular transporter elements include lipidsand liposomes, steroids, polyamines, nanotubes, nanoparticles,dendrimers (including guanidinium terminated dendrimers),cell-penetrating peptides, protein-transduction domains amino acids,peptoids (N-substituted glycines), and oligogcarbamates. Particular noteis made of the nuclear transcription activator protein (Tat) which isencoded by HIV type 1. Certain proteins contain subunits that arebelieved to enable their active translocation across the plasma membraneinto cells. In the specific case of HIV-1, this subunit is the basicdomain Tat(49-57) (RKKRRQRRR) (SEQ ID NO: 4), the “Tat nonamer.” Arg₇below in Structure 3 disclosed as SEQ ID NO: 3.

Also noted as useful molecular transporters are arginine oligomers ofabout 6-20 units (SEQ ID NO: 2) with particular note of, 7 to 15, andmore particularly 8 mer units, D-arginine oligomers, spaced arginineoligomers, and N-arginine peptoids as well as the examples of Table 1.Further noted are oligoarbamate transporters, and tetrameric dendrimers(e.g., Structure 4). Also noted are releasable-luciferin-transporterconjugates.

TABLE 1 Cell-penetrating peptides commonly used for deliveryapplications Cell- penetrating peptide Amino acid sequence PolyargininesRRRRRRRRR (R₉) (SEQ ID NO: 5) Tat₄₉₋₅₇ RKKRRQRRR (SEQ ID NO: 4)Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 6) (Antennapedia) Pep-1KETWWETWWTEWSQPKKKRKV(SEQ ID NO: 7) TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 8) Nuclear VQRKRQKLMP (SEQ IDNO: 9) localization sequences SKKKKIKV (SEQ ID NO: 10) GRKRKKRT (SEQ IDNO: 11)

Structure 5 shows reversible inhibitor of at least one neuron-specificPDZ domain which incorporates into the cellular endosome, where R is amolecular transporter (with or without a linker amino acid(s) withparticular reference to lysine. R₁ is at least about one amino acidcovalently bound, and in some embodiments R₁ is 2, 3, 4, 5, or 6 aminoacids. R₂ is isoleucine, leucine, alanine, phenylalanine, and valine. Ina specific embodiment, R₁ is β-alanine and R2 is valine. In someembodiments the P⁻⁴ position is usefully occupied by lysine, asparticacid, glutamic acid and arginine. As shown in Structure 5, the peptidechain extends to P⁻⁶ position.

Conventional Solid-Phase Synthesis Synthesis of 1[(N-Dde)-c[-Lys-Thr-Glu((βAla)-]-Val]

Step I.

Val-NovaSyn TGA resin (n mmol) was loaded into a peptide synthesisreaction vessel. The resin was suspended and swelled (45 min) withshaking using dichloromethane (DCM).

Step II.

Deprotection of the Fmoc group was carried out by adding piperidine-DMF(20% v/v; 10× resin volume) for 10 minutes. Then, piperidine-DMF wasdrawn off and the resin was treated with fresh piperidine-DMF (20% v/v;10× resin volume) for another 10 min while shaking. The solvent wasdrawn off, and the resin was washed with DMF (10× resin volume).

Step III.

Amino acid precursor (3 equiv), DIPCI (4 equiv), HOBt (6 equiv) and DMF(5× resin volume) were mixed within the reaction vessel, and were shakengently. After 2 h, a small amount of resin sample was removed, placed ina test tube and washed sequentially with DMF (3×1 mL), DCM (3×1 mL) andether (2×1 mL). The washed sample was submitted to the Kaiser Test. Anegative result was indicative of complete coupling.

Step IV.

If the reaction is not complete, additional DIPCI (1 equiv) was addedand the vessel shaken for another 2 h. If the coupling was complete, thesolution was drawn off and the resin was washed with DMF (10× resinvolume).

Steps I-IV

These protocols were used to synthesize the peptides KTEV (SEQ ID NO:12) using Dde-Lys(Fmoc)OH and Fmoc-Glu(PhiPr)OH for the P⁻³ and P⁻¹positions, respectively (Scheme 1). The cyclization was accomplished asfollows. Using step II, the lysine side chain Fmoc group was removed.Using step III in the linear peptide synthesis protocol, Fmoc-β-alanine(linker) was coupled to the side chain amino group of Lys. The PhiPrprotecting group from glutamate was removed using cleavage solution (5×resin volume, TFA/EDT/thioanisole/anisole/DCM (2:4:1:1:92 v/v)) for 30min. The solution was drawn off and the resin was treated with freshcleavage solution for another 30 min. This procedure was repeatedanother two times and finally washed with DMF (10×10× resin volume). Amalachite green test was carried out to confirm the presence of freeCOOH in the resin. Green beads are indicative of free COOH groups. Usingstep II, the Fmoc group on β-alanine was removed. An aggregation ofbeads was seen since there is a possibility of forming salt bridges orionic interactions between the free amino groups and carboxylic acidgroups. Then, the resin was washed three times with DIPEA in DMC (10×resin volume, 1:9 v/v; 3 min each time) to wash away any tracepiperidine. Finally, the resin was washed with DMF (10× resin volume).This step was followed by the addition of solvent mixtureDMSO-N-methylpyrrolidone (NMP) (10× resin volume, 1:4 v/v), which wasadded to the reaction vessel and gently shaken until the aggregatedresin spread out and was evenly suspended in solvent. HBTU (3 equiv),DIEPA (6 equiv), and HOBT (3 equiv) were then added and the vesselshaken gently (Scheme 1).

After 2 h, a small amount of resin sample (4-5 mg) was removed andplaced in a small test tube, and Kaiser's Test was carried out to checkthe reaction progress. A negative Kaiser's test was indicative ofcomplete cyclization. If the reaction was complete, the resin was washedwith DMF (10×10× resin volume). If the reaction was not complete, thesolvent was drawn off and fresh DMSO-N-methylpyrrolidone (NMP) (10×resin volume, 1:4 v/v) was added with HBTU (3 equiv), DIEPA (6 equiv),and HOBT (3 equiv) and the cyclization was repeated (Scheme 2). Whetherthe reaction was finished or not in the allocated time, the resin waswashed with DMF (10×1 mL) in order to avoid epimerization. Finally theresin was washed with DCM (5×10× resin volume). Then, a small amount ofresin sample (˜20 mg) was removed and resin cleavage solution (5× resinvolume, TFA/TIS (triisopropylsilane)/thioanisole/anisole (92:4:2:2,v/v)) was added with shaking for 2 h to obtain compound 1 (Scheme 1).The characterization for compound 1 is shown in Table 2.

TABLE 2 Compound 1 cyclic peptide synthesis and characterization.Peptide No. Structure Characterization 1

(N-Dde)-c[-Lys-Thr-Glu(βAla)-]-Val C₃₃H₅₂N₆O₁₀ Predicted [M+H]⁺: 694.0ESI-MS found: [M+H]⁺ 693.0, [M+Na]⁺ 715.0

Synthesis of 3 Cys-Lys-Asn-Tyr-Lys-c[-Lys-Thr-Glu(βAla)-]-Val

When the cyclization was completed as described for the synthesis of 1,the extension of the peptide was carried out by first removing the Ddeprotecting group from the N-terminus lysine (Scheme 2). Thisdeprotection was affected by a deprotection solution (10× resin volume,hydrazine/DMF (3% v/v), 10 min). After 10 min solution was drawn offwith a weak vacuum and fresh deprotection solution (10× resin volume,hydrazine/DMF (3% v/v), 10 min) was added while shaking. The Dde groupremoval procedure was repeated two more times, followed by washing withDMF (10×10× resin volume). The Kaiser's test was performed to confirmDde removal. A positive result was indicative of the free amino group.Steps II and III were repeated until the desired peptide was synthesizedwhich involved coupling of Fmoc-Lys(Boc)-OH, Fmoc-Tyr(OtBu)-OH,Fmoc-Asp(Trt)-OH, Fmoc-Lys(Boc)-OH and Fmoc-Cys(Trt)-OH. Step II wasused to remove the Fmoc group from the N-terminus. Then, a small amountof resin sample (˜20 mg) was removed before and after Fmoc-Cys(Trt)-OHcoupling and resin cleavage solution (5× resin volume, TFA/TIS(triisopropylsilane)/thioanisole/anisole (92:4:2:2, v/v)) was added withshaking for 2 h to obtain compound 2 and 3 (Scheme 2). Thecharacterization for compound 2 and 3 are shown in Table 3.

The solution was collected and separated equally into two 10 mL testtubes, followed by addition of ethyl ether (−20° C.) to reach 80% of thetotal tube capacity. The solution was mixed thoroughly and peptideprecipitate formed immediately. After centrifugation (8 min, 6000 rpm),the supernatant was decanted, fresh ether was added, and the pelletedpeptide was thoroughly mixed before another round of centrifugation(repeated 3 additional times).

TABLE 3 Compounds 2 and 3 cyclic peptide synthesis and characterization.Peptide No. Structure Characterization 2

Lys-Asn-Tyr-Lys-c[-Lys- Thr-Glu(βAla)-]-Val C₄₈H₇₉N₁₃O₁₄ Predicted[M+H]⁺: 1063.0 MALDI-TOF-MS found: [M+H]⁺ 1062.6, [M+2Na]⁺ 1107.7 3

Cys-Lys-Asn-Tyr-Lys-c[-Lys- Thr-Glu(βAla)-]-Val C₅₁H₈₄N₁₄O₁₅S Predicted[M+H]⁺: 1166.0 MALDI-TOF-MS found: [M+H]⁺ 1165.4 HPLC separationcondition: water (in 0.1% TFA): methanol = 80:20

Finally, the peptide was dissolved in distilled water (5-10 mL), frozen,and lyophilized for 24-48 h until a white powder was obtained. Peptideswere purified by RP-HPLC, and molecular masses confirmed by ESI andMALDI-TOF mass spectroscopy. A small amount of the crude peptide (1 mg)was submitted for mass spectral analysis (ESI-MS and/or MALDI-TOF massanalysis). Each peptide was purified by RP-HPLC (Phenomenex column,4.6×250 mm, pore size 1 Å). A 3 mg/mL solution of peptide sample wasthen prepared for analytical scale HPLC. Following the determination ofan appropriate solvent system, a peptide sample was prepared (50 mg/mLin water) and submitted to preparative scale HPLC. Thepeptide-containing fractions were identified and combined, followed bylyophilization. Another analytical scale HPLC was performed after allruns were collected; here all the fractions with peptide were combinedand an aliquot of the mixed solution (e.g., 1-5 μL of a 1 mg/mLsolution) was injected on to the column under the same conditions toconfirm the purity of the isolated peptide. If the fraction showed morethan one peak, a repeat of the purification procedure was carried out.

Synthesis of 4 & 5 Synthesis of 4[(N-Fmoc)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-CONH₂] (SEQ ID NO: 3)

The procedure for this peptide was similar to the general linear peptidesyntheses, which was manually prepared and purified using standardFmoc-based solid-phase peptide synthesis protocols. The synthesis ofoligoarginine was a particularly difficult synthetic procedure comparedto standard linear peptide synthesis. The synthesis was carried out onRink Amide AM resin (n mmol). The coupling steps were more difficult,and following the coupling of several arginine residues, the beadsaggregate after Fmoc-deprotection. The coupling protocol was modified tosolve the problem by using pre-activated solutions of arginine.

Coupling of Arginine.

Pre-activation was accomplished by combining DIPEA (4 equiv), HOBT (2.5equiv), HBTU (2.5 equiv) and DMF (5× resin volume), which were shakengently for 7 min. Arginine precursor (3 equiv) was then added. After 2h, a small amount of resin sample (4-5 mg) was removed and submitted tothe Kaiser Test. A negative result was indicative of complete coupling.After the coupling was complete, the reaction solution was drawn off andresin was washed with DMF (10×10× resin volume). Fmoc group removal waseffected by the standard procedure, followed by washing with DCM toremove trace DMF (10×10× resin volume). Coupling of arginine was carriedout another six times. Small amount of resin beads were cleaved off toconfirm the synthesis of compound 4. The cleavage was carried outsimilar to standard protocols using resin cleavage solution (5× resinvolume, TFA/TIS (triisopropylsilane)/anisole (92:6:2, v/v)) aftershaking for 28 h. The characterization for compound 4 is shown in Table4.

TABLE 4 Compound 4 poly-arginine transporter synthesis andcharacterization. Peptide No. Structure Characterization 4

(N-Fmoc)-Arg-Arg- Arg-Arg-Arg- Arg-Arg-CONH₂ (SEQ ID NO: 3) C₅₇H₉₇N₂₉O₉Predicted [M+H]⁺: 1333.5 MALDI-TOF-MS found: [M+H]⁺ 1332.8, [M-156]⁺1176.5, [M-312]⁺ 1020.5 Synthesis of 5[Cys(Npys)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-CONH₂] (SEQ ID NO: 13). Thisprocedure was the same as the synthesis of 5, but included an additionalcoupling of Boc-Cys(Npys)-OH or Fmoc-Cys(Npys)-OH to the N-terminususing general coupling protocols. The characterization for compound 5 isshown in Table 5.

TABLE 5 Compound 5 poly-arginine transporter synthesis andcharacterization. Peptide No. Structure Characterization 5

Cys(Npys)-Arg-Arg- Arg-Arg-Arg- Arg-Arg-CONH₂ (SEQ ID NO: 13)C₅₀H₉₄N₃₂O₁₀S₂ Predicted [M+H]⁺: 1367.5, [M+H-(Npys)]⁺ 1213.5 ESI-MSfound: [M+H-(Npys)]⁺ 1213.5, [M+H-(Npys)-156]⁺ 1057.5, [M+H-(Npys)-312]⁺901.0

Synthesis of 6 (Structure 1)

The synthesis was carried out by disulfide coupling of the on-beadoligoarginine compound 5 with previously prepared macrocyclic compound 3(˜1.1 equiv). The coupling was performed in 0.1% TFA in DMF (20× resinvolume). The orange solution slowly developed a bright yellow and themixture was permitted to stir overnight (14 h) at room temperature. Thebright yellow color formation is a confirmation of the Npys (cystineside chain protection) removal in the reaction (FIG. 1). Afterexhaustive washing steps with DMF, DCM, MeOH, DMSO and ethyl ether(which should remove all the unreacted macrocyclic compound 3), theligand was cleaved off the beads using cleavage cocktail (5× resinvolume, TFA/TIS (triisopropylsilane)/anisole (92:6:2, v/v)) with shakingfor 6 h. The lyophilized product of the final product was crystalline,which was a completely different than the white power that was obtainedfor macrocyclic compound 3. The characterization for compound 6 is shownin Table 6.

TABLE 6 Structure 1 characterization. Peptide No. and Code Structure andCharacterization 6

  Arg₇ above disclosed as SEQ ID NO: 3CONH₂-(Arg)₇-Cys-Cys-Lys-Asn-Tyr-Lys-c[-Lys-Thr-Glu(βAla)-]-ValC₉₆H₁₇₄N₄₄O₂₃S₂ Predicted [M+H]⁺: 2378.0 MALDI-TOF-MS found: [M+H]⁺2384.5, [M+H-156]⁺ 22290.0, [M+H-312]⁺ 2073.0, [M-cyclic]⁺ 1213.4,[M-(C-R₇)Na]⁺ 1164.4 [M-cyclic+Na-H]⁺ 1235.5 HPLC separation condition:water (in 0.1% TFA): methanol = 75:25

Synthetic Protocol for Structure 1: Microwave Solid-Phase Synthesis

Structure 1 was synthesized on Rink amide AM (0.6 mmol/g, Novabiochem)resin (cat no. 01-64-0038) using microwave solid phase peptide synthesisprotocols developed in our lab. The synthesis was carried out bydisulfide coupling of the on-bead oligoarginine compound 5 withpreviously prepared macrocyclic compound 3 (˜1.1 equiv).

Synthesis of 3 Cys-Lys-Asn-Tyr-Lys-c[-Lys-Thr-Glu(βAla)-]-Val

The macrocyclic compound 3 was synthesized on Fmoc-Val NovaSyn TGA resin(N mmol, 0.2 mmol/g, cat. no. 04-12-2671). After the initial Fmocdeprotection using 25% piperidine in DMF MW, 70° C., 6.5 min, couplingof Fmoc-Glu(PhiPr)-OH was carried out using 5 equiv Fmoc-Glu(PhiPr)-OH,5 equiv HBTU in DMF, MW, 70° C., 8 min followed by Fmoc deprotectionusing 25% piperidine in DMF MW, 70° C., 6.5 min as previously described.Then Fmoc-Thr(OtBu)-OH and Dde-Lys(Fmoc)-OH one after another using 5equiv Fmoc-aa-OH, 5 equiv HBTU in DMF, MW, 70° C., 8 min for amino acidcoupling and 25% piperidine in DMF MW, 70° C., 6.5 min, for Fmocdeprotection. After the Fmoc deprotection of lysine side chain,Fmoc-βAla-OH was coupled using 5 equiv Fmoc-βAla-OH, 5 equiv HBTU inDMF, MW, 70° C., 8 min. This step was followed by PhiPr groupdeprotection of glutamate side-chain using 2% TFA, 2% triisopropylsilane, 2% triethylsilane in 1,2 dichloromethane, 70° C., 15 min.following PhiPr group deprotection, the N-terminal βAla Fmoc group wasdeprotected using 25% piperidine in DMF MW, 70° C., 6.5 min. Thenon-bead cyclization followed using 3 equiv HBTU, 3 equiv HCTU, DMF, MW,70° C., 12 min. After the cyclization the extension of the peptide wascarried out by first removing the Dde protecting group from theN-terminus lysine. The Dde group was deprotected using 5% hydrazinemonohydrate in DMF MW, 70° C., 7.5 min. This step was followed bycoupling of Fmoc-Lys(Boc)-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Asp(Trt)-OH,Fmoc-Lys(Boc)-OH and Fmoc-Cys(Trt)-OH using 5 equiv Fmoc-aa-OH, 5 equivHBTU in DMF, MW, 70° C., 8 min for coupling step followed by Fmocdeprotection using 25% piperidine in DMF MW, 70° C., 6.5 min. Then thebeads were thoroughly washed with DMF, DCM, ethyl ether. Finally theside chain deprotection and resin cleavage was effected by treating with2% anisole, 2% thioanisole, 2% triisopropyl silane, 94% TFA, MW, 40° C.for 30 min. Compound 3 was purified by RP-HPLC, and molecular massesconfirmed by MALDI-TOF mass spectroscopy.

Synthesis of 5 [Cys(Npys)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-CONH₂] (SEQ ID NO:13)

The compound 5 was synthesized on Rink amide AM (0.6 mmol/g,Novabiochem) resin (cat no. 01-64-0038) using microwave solid phasepeptide synthesis protocols. The initial Fmoc deprotection using 25%piperidine in DMF MW, 70° C., 6.5 min, was followed by coupling ofFmoc-Arg(Pbf)-OH was carried out using 5 equiv Fmoc-Arg(Pbf)-OH, 5 equivHBTU in DMF, MW, 70° C., 10 min followed by Fmoc deprotection using 25%piperidine in DMF MW, 70° C., 6.5 min. The coupling of Fmoc-Arg(Pbf)-OHwas repeated another six times to give theFmoc-Arg(Pbf)-(Arg(Pbf))₆-on-bead (SEQ ID NO: 14). This was followed bythe N-terminal Fmoc deprotection of Arginine followed by coupling ofBoc-Cys(Npys)-OH using 5 equiv Boc-Cys(Npys)-OH, 5 equiv HBTU in DMF,MW, 60° C., 8 min. The synthesis of compound 5 was carried out prior tothe final on-bead disulfide coupling to give compound 6 (Structure 1).

Synthesis of Compound 6 (Structure 1)

The synthesis was carried out by disulfide coupling of the on-beadoligoarginine compound 5 with previously prepared macrocyclic compound 3(˜1.1 equiv). Boc-Cys(Npys)-(Arg(Pbf))₇-on-bead (SEQ ID NO: 15) (N mmol)resin was first thoroughly washed with DMF, DCM, methanol, ethyl ether.Then a solution was prepared using ˜N mmol of compound 3 in 0.1% TFA inDMF and added to Boc-Cys(Npys)-(Arg(Pbf))₇-on-bead (SEQ ID NO: 15). Thenthe mixture was reacted for 12 min, MW, 70° C. The mixture was allowedto come to room temperature while shaking and allowed to shake foranother 1 hr. The orange solution developed a bright yellow mixture atthis stage indicative of the Npys (cystine side chain protection)removal in the reaction. After exhaustive washing steps with DMF, DCM,MeOH, DMSO and ethyl ether, the ligand was cleaved off the beads usingcleavage cocktail 2% anisole, 2% thioanisole, 2% triisopropyl silane,94% TFA, MW, 40° C. for 45 min. Compound 6 was purified by RP-HPLC, andmolecular masses confirmed by MALDI-TOF mass spectroscopy.

One aspect of the present invention usefully employselectrophysiological screening of Structure 1. Particular attention isdrawn to an electrophysiological assay that rapidly screens forcompounds which are inhibitors that target neuron-specific PDZ domains.Recordings of cellular activity were made from acute slices of ratretinal and brain tissue to examine the effects of Structure 1 onsynaptic Kainate/AMPA, NMDARs and to test for protection againstexcitotoxic insults. Kainate/AMPA and NMDAR components weredistinguished pharmacologically. Noted are the selective AMPA-antagonistNBQX and the competitive NMDA-antagonist CPP. A multi-electrode arraywas used that enabled examination of activity from sixty neuronssimultaneously. This array addressed whether a test compound providedneuroprotection against kainate or N-methyl-D-aspartic acid(NMDA)-induced death. Briefly, slices were perfused for prolongedperiods (about 30 min) with NMDA or kainate agonists until activity inhalf of the neurons is lost and responses cannot be rescued by washingout the NMDA or kainate (assumed to be resulting from neuronal death).Pretreatment with the compound of Structure 1 (30 uM) or cotreatmentwith the compound of Structure 1 and an NMDA receptor agonist providedefficacy and potency in preventing neuronal death. In a test, this doseof 30 uMolar prevented cell death.

In addition to the retinal tissue as a model for Stroke, compounds ofthe present invention were tested in hippocampal neurons by deliveringan excitotoxic insult, commensurate with a Stroke, which resulted in thepermanent loss of functional activity in these cells. This loss wasprevented by the co-application of the compound of Structure 1. Resultswere confirmatory of the retinal data and extends it into tissues thatare more strongly associated with CNS disorders such as Stroke andneurodegenerative diseases. Noted is therapeutic treatment for autismdisorders and cognitive decline symptomatology conditions.

The compounds of the present invention were further tested on synapticplasticity and in particular LTP (long-term potentiation) in thehippocampus. Data indicated enhanced induction and magnitude of LTP. Itis noted that LTP is widely considered to be the basis for learning andmemory and enhancements and deficits in LTP have been reported ascorrelated with increased and decreased performance in behavioral teststhat measure these phenomena, respectively.

Attention is drawn to pretreatment by administration of atherapeutically effective amount of a composition of Structure 5 about48 to about ½ hour in advance of neuro-stress, and particularly aboutwithin 6 hrs of the neuro-stress event. For post neuro-stress treatmentadministration as soon as possible is noted. Administration within 30minutes to one hour from onset of neuro-stress is particularly noted.Noted are single doses from about 0.1 uM to about 100 uM, or continuousinfusions of 0.01-5 μM/hr via intrathecal, intraventrical or intravitralroutes.

Investigation further included monitoring the selective uptake of thepoly-arginine (Structure 3)-and the mysterolated-tailed PDZ-mimic(e.g.,Structure 1 and 2) tagged with a fluorescent marker crossed thevitreal/retina barrier when administered intravitrealy and was shown toreadily cross the pia mater/brain barrier and localize in neurons deepto these barriers within the retina, spinal cord and brain.

Uptake in the retina was observed as follows. Retinas exposed to 1.5-6nmol of Structure 1 compound showed about a 50% uptake pattern, 6hrs-following intravitreal injection. In numerous populations of retinalneurons located in both the ganglion cell (GCL) and inner nuclearlayers. Structure 1 compound also selectively accumulated withinganglion cell axons as evident by their intense labeling at the opticnerve head. These results provide evidence that an intravitrealinjection of Structure 1 reaches neurons that are sensitive to glutamateexcitotoxicity within the retina and axonal fibers that are unsheathedby mylenated covering within the optic nerve, which are also known sitessensitive to glutamate excitotoxicty.

Example 1 Intrathecal Injection

Intrathecal injection of Structure 1 compound in rats was tested. Ratswere prepped for intrathecal injection containing 16 nmol of Structure 1compound at the level of CV2. 24 hrs following the injection the ratswere perfused with 4% PFA and frozen cross-sections of the cord weremounted on glass slides and photographed. Structure 1 compositioncrosses the pia mater/brain barrier and selectively accumulates inneurons located within the gray matter of the cord. Uptake of Structure1 compound occurred in the cord at the site of the injection (highcervical region of the cord), and was shown to label neurons within thegray area from high cervical, all the way down to the conus medularis.In addition, the labeled peptide was also shown to accumulate in cellslocated in the cortex, hippocampus, brainstem and striatum with thesecervical injections. These data indicate that intrathecal delivery ofStructure 1 is useful to preload all spinal cord neurons with thePDZ-mimic and provide neuroprotection.

The compound of this invention are useful in the therapeutic treatmentof neurological insult such as stroke, traumatic brain injury, epilepsyas well as for pain and neurodegenerative disease (collectively,“neuro-stress”). Particular note is made of the prophylactic treatmentof neuro-stress by administering a therapeutic dosage in advance ofinsult. This method is available, for example, prior to a surgicalprocedure that would invade the brain. Without being bound by anyparticular theory it is believed stroke and other neurological insultsentail brain cells being starved of oxygen, glucose, nutrients and anassociated build up of waste materials, that include glutamate, leadingto an excitotoxic insult. The neurons of the stroke patient becomehyper-excitable due to the release of large concentrations of glutamate(10-fold greater than basal) which activate receptors includingextrasynaptic NMDA receptors). This leads to the gating of large amountsof calcium though these normally quiet receptors. The intracellularcalcium concentration rises from basal levels (0.05-0.2 uM) to a level(luM) that triggers the activation of cell death pathways and the resultis brain damage to the affected areas of the brain. Compounds of thepresent invention are usefully administered at therapeutic doses withinabout 30 minutes to 1 hour. In some instances an oral formulation, anasal spray or an acute injection given i.v. or intrathecally to inducea therapeutic concentration in the brain is indicated. This protectsneural tissue. Particular note is made of drug administration which, attherapeutic levels, interferes with the signal mediated by the risinglevels of calcium so that the elevated calcium does not trigger the celldeath pathway. As the ischemic condition abates and calcium levelsreturn to normal the patient recovers and brain damage has been averted.

Example 2 Therapeutic Protocol in Stroke

A 48 year old female presents with an ischemic episode resulting suddennumbness the face, and left arm, confusion, and trouble speaking. Within2 hours of onset, Structure 1 composition is administered at 50 mg/Kgi.v. Within 48 hours the patient recovers without significant braindamage. Doses of from about 1 to about 100 mg/Kg are noted.

The compositions of this invention possess valuable pharmacologicalproperties. They are useful in the treatment of stroke or anoxic braininjury. This effect can be demonstrated, for example, using the methodof prompt administration upon neurological insult. Administration iscontemplated to include chronic, acute or intermittent regimens.

The pharmacologically active compositions of this invention can beprocessed in accordance with conventional methods of Galenic pharmacy toproduce medicinal agents for administration to patients, e.g., mammalsincluding humans.

The compositions of this invention can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, or enteral (e.g.,oral or inhalation) use which do not deleteriously react with the activecompositions. Suitable pharmaceutically acceptable carriers include butare not limited to water, salt solutions, e.g., saline. Thepharmaceutical preparations can be sterilized and if desired mixed withauxiliary agents, e.g., 1 salts for influencing osmotic pressure,buffers and the like which do not deleteriously react with the activecompositions. They can also be combined where desired with other activeagents, e.g., TPA (Tissue Plasminogen activator).

In some embodiments of the present invention, dosage forms includeinstructions for the use of such compositions.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Ampulesare convenient unit dosages.

Sustained or directed release compositions can be formulated, e.g.,liposomes or those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. It is also possible to freeze-dry the newcompositions and use the lyophilizates obtained, for example, for thepreparation of products for injection.

Generally, the compositions of this invention are dispensed in unitdosage form comprising about 1 to about 100 mg in a pharmaceuticallyacceptable carrier per unit dosage.

The dosage of the compositions according to this invention generally are1 to 100 mg/kg/day, preferably 1 to 10 (especially if the general dosagerange spans an order of magnitude or more), when administered topatients, e.g., humans to treat (e.g., cardiac insufficiency)analogously to the known agent (hydrochlorothiazide (HydroDIURIL®), andis to 25-50 mg daily TID mg/kg/day when administered to treat(hypertension); (repeat for all activities and indications).Alternatively, treat as an IV bolus, then IV infusion similar toThrombolytic agents such as alteplase (TPA). or antiarrhythmic drugse,g, atenolol (IV 50 10 mg) or anti-Parkinson drugs benztropine(Congentin®) IV 1-6 mg daily, or entacapone 200-1,600 mg.

It will be appreciated that the actual preferred amounts of activecompositions in a specific case will vary according to the specificcompositions being utilized, the particular compositions formulated, themode of application, and the particular situs and organism beingtreated. Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject compositions and of a known agent, e.g., bymeans of an appropriate, conventional pharmacological protocol.

1. A composition which is reversible inhibitor of at least oneneuron-specific PDZ domain comprising

wherein R is a molecular transporter with or without a linker aminoacid; R₁ is at least about one amino acid covalently bound; and, R₂ isisoleucine, leucine, alanine, phenylalanine, or valine.
 2. Thecomposition of claim 1 wherein R₁ is β-alanine.
 3. The composition ofclaim 1 wherein R₂ is valine.
 4. The composition of claim 1 wherein theP⁻⁴ position is lysine, aspartic acid, glutamic acid or arginine.
 5. Thecomposition of claim 1 having the structure:


6. The composition of claim 1 having the structure:


7. The composition of claim 1 wherein R comprises a liposome, steroid,polyamine, nanotube, nanoparticle, dendrimer, cell-penetrating peptide,protein-transduction domain amino acid, peptoid, (N-substitutedglycine), oligogcarbamate, arginine oligomer of about 6-20 units (SEQ IDNO: 2), D-arginine oligomer, spaced arginine oligomer, N-argininepeptoid, oligoarbamate transporter, or tetrameric dendrimer.
 8. Theinhibitor of claim 1 wherein R comprises (SEQ ID NO: 3):


9. A method of treatment of neuro-stress comprising administering to asubject a therapeutically effective amount of the composition ofclaim
 1. 10. The method of claim 9 wherein the neuro-stress is selectedfrom the group comprising stroke, traumatic brain injury, epilepsy, painor neurodegenerative disease.
 11. The method of claim 9 wherein saidadministration is in advance of said neuro-stress.
 12. The method ofclaim 9 further compromising cotreatment with a therapeuticallyeffective amount of an NMDA receptor agonist.
 13. The method of claim 12wherein said NMDA receptor agonist is selected from the group comprisingAMPA, kainate, ketamine and NMDA.
 14. The method of claim 9 wherein saidtherapeutic effective dose of the composition of claim 1 is from about0.1 μM to about 100 μM.
 15. The method of claim 14 wherein said dosageis from about 20 μM to about 40 μM.
 16. The method of claim 9 whereinadministration is parenteral, oral, buccal, sublingual, or by nasalspray.
 17. The method of claim 16 wherein administration is intrathecal.18. The method of claim 13 wherein said receptor agonist is ketaminefrom about 10 to about 250 mg.